Abstract
During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.
Highlights
During DNA replication in eukaryotic cells, short singlestranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand
A Flap-like Structure Is Observed by electronic microscopy (EM) in Replication Forks—To determine whether the flap pathway is used by eukaryotic cells to remove RNA-DNA primers from Okazaki fragments (Fig. 1A), we first examined whether flap structures are formed in the replication forks in wild type (WT) fission yeast cells
Flap Structures Significantly Increased in Replication Forks of fen1Ϫ, dna2Ϫ, and fen1Ϫ-dna2Ϫ Cells Compared with WT Cells—we examined the flap structures in replication forks from fen1Ϫ, dna2Ϫ, and fen1Ϫ-dna2Ϫ cells
Summary
During DNA replication in eukaryotic cells, short singlestranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. 2 The abbreviations used are: pol, polymerase; nt, nucleotide(s); BND-cellulose, benzoylated naphthoylated diethylaminoethyl cellulose; RPA, replication protein A; ssDNA, single-stranded DNA In Escherichia coli and bacteriophage T4 and T7, the short RNA primers at the 5Ј-end of Okazaki fragments are hydrolyzed directly by the 5Ј- to 3Ј-exonuclease activity of DNA pol I, RNase H, and T7 gene 6 DNA exonuclease, respectively (9 –11). Budding yeast Saccharomyces cerevisiae cells are viable when both the Fen and RNase H2 genes are deleted [12] This observation suggests either that S. cerevisiae cells possess redundant RNA and DNA exonucleases for primer digestion or that the exonuclease pathway is not a major pathway in the primer removal event.
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